Abstract

Recently, we have proposed a novel strategy for a cell-specific gene therapy system
based on responses to intracellular signals. In this system, an intracellular signal
that is specifically and abnormally activated in the diseased cells is used for the
activation of transgene expression. In this study, we used protein kinase C (PKC)α
as a trigger to activate transgene expression. We prepared a PKCα-responsive polymer
conjugate [PPC(S)] and a negative control conjugate [PPC(A)], in which the phosphorylation
site serine (Ser) was replaced with alanine (Ala). The phosphorylation for polymer/DNA
complexes was determined with a radiolabel assay using [γ-32P]ATP. PPC(S)/DNA complexes were phosphorylated by the addition of PKCα, but no phosphorylation
of the PPC(A)/DNA complex was observed. Moreover, after microinjection of polymer/GFP-encoding
DNA complexes into HepG2 cells at cation/anion (C/A) ratios of 0.5 to 2.0, significant
expression of GFP was observed in all cases using PPC(S)/DNA complexes, but no GFP
expression was observed in the negative control PPC(A)/DNA complex-microinjected cells
at C/A ratios of 1.0 and 2.0. On the other hand, GFP expression from PPC(S)/DNA complexes
was completely suppressed in cells pretreated with PKCα inhibitor (Ro31-7549). These
results suggest that our gene regulation system can be used for tumor cell-specific
expression of a transgene in response to PKCα activity.